Role of the kinesin neck linker and catalytic core in microtubule-based motility.

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].

The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Change of intradisc pressure versus volume change.

To gain further insight into the biomechanics of the intervertebral disc and determine a potential mechanism for causation and relief of symptoms related to a herniated disc, the pressure-volume relationship was determined within the nucleus pulposus (NP). In 17 intact human cadaver lumbar discs, pressure was measured continuously within the NP by means of a miniature strain gauge at the tip of a size 4 French (1.3 mm) catheter inserted into the NP. The volume of the NP was increased at the slow, continuous rate of 0.034 ml/min by the pump-regulated infusion of saline colored with methylene blue. In 12 unloaded discs, NP pressure rose in a linear fashion (linear r2 = 0.96) from an initial mean pressure of 174 +/- 81 kPa. The mean rate of pressure rise was 327 +/- 109 kPa/ml of volume increase. The peak pressure reached was limited to 550 kPa by the capacity of the strain gauge. Similar linear relationships were obtained during saline infusion into 5 vertically loaded discs. The data define the pressure-volume relationship within the disc and show that the NP, surrounded by the relatively inelastic annulus and the solid vertebral end-plates, has the properties of a tight hydraulic space in which a large pressure rise will regularly result from a small increase in volume. Presumably, the opposite is also true. The data may provide a biomechanical basis for variation in symptoms related to physiological changes in disc volume, and for any benefit obtained from interventions designed to remove disc tissue.

Laser radiation at various wavelengths for decompression of intervertebral disk. Experimental observations on human autopsy specimens.

The interaction of laser radiation with the nucleus pulposus from autopsy specimens of human intervertebral disks was evaluated at different wavelengths (193 nm, 488 nm & 514 nm, 1064 nm, 1318 nm, 2150 nm, 2940 nm, and 10600 nm). A significant correlation of linear least squares fit of the mass ablated as a function of incident energy was found for all lasers used except the Excimer at 193 nm. The 2940-nm Erbium:YAG laser was most efficient in terms of mass of disk ablated per joule in the limited lower range where this wavelength was observed. At higher energy levels, the CO2 laser in the pulsed mode was most efficient. However, the Nd:YAG 1064-nm and 1318-nm lasers are currently best suited for percutaneous laser disk decompression because of the availability of usable waveguides. Carbonization of tissue with the more penetrating Nd:YAG 1064-nm laser increases the efficiency of tissue ablation and makes it comparable to the Nd:YAG 1318-nm laser.

Experimental Nd:YAG laser disintegration of methylmethacrylate. Analysis of gaseous products.

The difficulty of removing intramedullary methylmethacrylate (MM) in revision of hip prostheses is considerable. To facilitate its removal from bone, Nd:YAG laser energy was delivered to a bovine anatomic specimen of the femur with the medullary canal filled with MM. Through an endoscope the laser was observed to soften and ablate the methyl methacrylate. The vapors generated by this process were analyzed by mass spectrometry. The two chief components were hydrogen and carbon monoxide, followed by carbon dioxide, methane, acetylene, and C3 unsaturated hydrocarbons. A high capacity smoke evacuator must be used in conjunction with laser vaporization of methylmethacrylate. The method should be evaluated under in vivo conditions in dogs before it is attempted in patients.

Association between transient pulmonary congestion during acute myocardial infarction and high incidence of death in six months.

In a prospective multicenter study of 866 patients after acute myocardial infarction (AMI), an increased or excessive mortality rate (13%) was confined to the first 6 months after AMI. In the subsequent 18 months of follow-up, the mortality rate (4%) was similar to that in coronary patients in chronic stable condition. Analysis of patients who died in the first 6 months revealed that 55% had had pulmonary congestion at the time of the index AMI. Neither these patients nor the others who died in the early period were found to have more severe ventricular dysfunction, more malignant arrhythmias or more severe ischemia than patients who died after 6 months. The reason for the high and early mortality in patients with pulmonary congestion is not clear, particularly because 30% had reasonable ventricular function, with an ejection fraction of more than 40%. However, given the poor prognosis of these patients, early and aggressive diagnostic efforts should be undertaken to exclude jeopardized regions remote from the initial AMI.

Vascular recanalization with the argon laser: the role of blood in the transmission of laser energy.

Successful revascularization of stenosed arteries with the argon laser optical fiber catheter requires delivery of high-power-density energy to the lesion. Because of the high energy absorption by hemoglobin at the argon laser wavelengths, which would attenuate transmission of laser energy to the obstruction, we measured argon laser transmission through blood at different concentrations. We found that no transmission occurred through 1 mm of whole blood with hematocrit of 42 and that blood must be diluted to a hematocrit below 1 for laser energy to be delivered. Successful use of the intravascular argon laser is dependent on the creation of a transmission medium by displacement of blood by saline or other non-absorbing substance.

Absence of distal emboli during in vivo laser recanalization.

We used an argon laser to recanalize occluded arteries in vivo and to determine the extent of any particulate matter resulting from the procedure that might cause embolization. Thrombosis was achieved by balloon de-endothelialization and thrombin injection in 12 canine femoral or carotid arteries in six dogs. The resulting totally occlusive thrombi (2.5-8.5 cm in length) were laser treated in situ 2-29 days after formation, using laser tip power outputs of 0.8-3.4 W. The treated arterial segment was perfused before, during, and after the recanalization procedure with 1,000 ml of heparinized saline. All saline passing through the artery was collected and studied by filtration through 20-micron pore filter paper; 50-ml unfiltered aliquots were studied by automated, calibrated cell counting and microscopically after centrifugation and staining. All vessels except one were completely recanalized. Filtration yielded a thin reddish, dust-like residue. No large fragments were found in 11 of 12 effluents. The residue after centrifugation was almost entirely composed of erythrocytes plus one to four 20-30-micron strands of amorphous cellular material per high-power field. Cell counting showed that 99.1% of the material in the effluents was smaller than 9.37 micron. These results demonstrate the presence of some filterable debris following laser recanalization of intraarterial thrombi, but it probably lacks any physiologic significance. Further study of this effect of laser recanalization is needed.

Complete patency in thrombus-occluded arteries two weeks after laser recanalization.

The potential problem of rethrombosis after laser recanalization was studied in 16 thrombus-occluded canine femoral arteries. Balloon de-endothelialization and thrombin-human blood injection produced adherent, completely occlusive thrombi 4.13 +/- 1.54 cm in length; laser exposure of the thrombi occurred at 18.35 +/- 22.1 hours. The argon laser catheter was introduced via a proximal arteriotomy and a power of 3.83 +/- 0.58 W delivered for 411.3 +/- 296.87 seconds. Follow-up period was 14 days. All arteries were patent immediately after and 14 days following lasing, as demonstrated by angiography. There was no vessel perforation. Seven of the dogs were maintained on aspirin and dipyridamole 4 days before and throughout the study, but there were no differences in thrombus length, laser power, or duration of laser exposure between these dogs and those receiving no anti-platelet therapy. Control thrombosed arteries (without laser energy application) showed no autolysis within 14 days in all dogs and up to 95 days in three dogs followed for this period of time. These data show that rethrombosis of totally occluded, thrombosed arteries is not present up to 2 weeks later after laser recanalization, with or without the aid of anti-platelet therapy.

Measurement of regional myocardial perfusion using collimated miniature radiation detectors and the method of 133-xenon washout.

A new approach to the measurement of local myocardial perfusion based on the application of highly-collimated miniature cadmium telluride radiation detectors to measure washout of 133-Xenon from well-defined tissue volumes is presented. Single-hole collimators with length/diameter ratios of 1 (L = 4 mm, D = 4 mm) and 4 (L = 12 mm, D = 3 mm) were employed as prototype designs. The probe field of view was characterized theoretically using a spherical model of the myocardium in conjunction with experimental point source response measurements for each collimator. Method evaluation using two medium resolution collimators (L/D = 1) was effected by performing left main and circumflex coronary artery perfusion studies at controlled but variable flows in the dog heart. An excellent correlation (r greater than 0.99) between actual and estimated perfusion determined from 65 washout curves over the flow range 0.38 to 3.18 ml/min/gm was demonstrated. The ability to resolve regional flow differences was verified by measuring tracer washout from circumflex and left anterior descending regions of the myocardium, using two high resolution collimators (L/D = 4), in a preparation where the left main coronary artery was cannulated and a snare was placed around the circumflex coronary artery to selectively reduce flow to that region.